- Email: [email protected]
Microtubule Assembly: Lattice GTP to the Rescue
Current Biology Vol 19 No 4 R174
6.
7.
8.
9.
10.
11.
environmental factor that regulates fat storage. Proc. Natl. Acad. Sci. USA 101, 15718–15723. Rakoff-Nahoum, S., Paglino, J., EslamiVarzaneh, F., Edberg, S., and Medzhitov, R. (2004). Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118, 229–241. Mazmanian, S.K., Liu, C.H., Tzianabos, A.O., and Kasper, D.L. (2005). An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 122, 107–118. Stappenbeck, T.S., Hooper, L.V., and Gordon, J.I. (2002). Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells. Proc. Natl. Acad. Sci. USA 99, 15451–15455. Turnbaugh, P.J., Ley, R.E., Mahowald, M.A., Magrini, V., Mardis, E.R., and Gordon, J.I. (2006). An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027–1031. Rakoff-Nahoum, S., and Medzhitov, R. (2007). Regulation of spontaneous intestinal tumorigenesis through the adaptor protein MyD88. Science 317, 124–127. Wen, L., Ley, R.E., Volchkov, P.Y., Stranges, P.B., Avanesyan, L., Stonebraker, A.C., Hu, C., Wong, F.S., Szot, G.L., Bluestone, J.A., et al. (2008). Innate immunity and intestinal microbiota in the development of Type 1 diabetes. Nature 455, 1109–1113.
12. Chen, G., Shaw, M.H., Kim, Y.G., and Nunez, G. (2008). Nod-like receptors: Role in innate immunity and inflammatory disease. Annu. Rev. Pathol., epub ahead of print. 13. Akira, S., Uematsu, S., and Takeuchi, O. (2006). Pathogen recognition and innate immunity. Cell 124, 783–801. 14. Watanabe, T., Asano, N., Murray, P.J., Ozato, K., Tailor, P., Fuss, I.J., Kitani, A., and Strober, W. (2008). Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis. J. Clin. Invest. 118, 545–559. 15. Bouskra, D., Brezillon, C., Berard, M., Werts, C., Varona, R., Boneca, I.G., and Eberl, G. (2008). Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis. Nature 456, 507–510. 16. Hasegawa, M., Yang, K., Hashimoto, M., Park, J.H., Kim, Y.G., Fujimoto, Y., Nunez, G., Fukase, K., and Inohara, N. (2006). Differential release and distribution of Nod1 and Nod2 immunostimulatory molecules among bacterial species and environments. J. Biol. Chem. 281, 29054–29063. 17. Boughan, P.K., Argent, R.H., Body-Malapel, M., Park, J.H., Ewings, K.E., Bowie, A.G., Ong, S.J., Cook, S.J., Sorensen, O.E., Manzo, B.A., et al. (2006). Nucleotide-binding oligomerization domain-1 and epidermal growth factor receptor: critical regulators of beta-defensins during Helicobacter pylori infection. J. Biol. Chem. 281, 11637–11648.
Microtubule Assembly: Lattice GTP to the Rescue Recent work describes the surprising finding that cellular microtubules have islands of GTP-bound tubulin within their lattices, in contrast to the longstanding view that all but the very tips of growing microtubules are made up entirely of GDP–tubulin. These GTP–tubulin islands may act as stop signs or speed bumps, switching a shortening microtubule back into a growing state. Lynne Cassimeris Microtubule polymers are far from static; each polymer continually and unpredictably shifts between growing and shortening states (Figure 1). This turnover, a process called dynamic instability, allows microtubules to reorganize swiftly in response to cues. Dynamic turnover is critical for a number of cell functions and several anti-cancer drugs halt cells in the midst of division by blocking microtubule dynamics [1]. The most puzzling mechanistic aspects of dynamic instability are the switches between the polymerization and depolymerization states because these are such rare events compared with the addition and removal of thousands or tens of thousands of subunits from a microtubule end before a switch occurs. A recent paper in Science [2] provides surprising evidence that GTP-bound tubulin, the type of tubulin
dimer thought to be present only at microtubule ends, is also scattered throughout the microtubule lattice. These lattice GTP–tubulin subunits may function to stimulate rescue when a shortening microtubule stops losing subunits and begins to polymerize again. The structure of the microtubule tip governs whether the tubulin subunits that form the microtubule polymer will add or subtract from the polymer’s end (Figure 1). Polymerizing microtubules are typically not blunt-ended tubes, but instead have sheet-like extensions of tubulin protofilaments (Figure 1) that eventually close to form a tube. Shortening microtubules look very different — their protofilaments are no longer straight and they lose contact with their neighbors. These shortening protofilaments peel away from the microtubule lattice and soon fall apart into individual tubulin subunits. In
18. Masumoto, J., Yang, K., Varambally, S., Hasegawa, M., Tomlins, S.A., Qiu, S., Fujimoto, Y., Kawasaki, A., Foster, S.J., Horie, Y., et al. (2006). Nod1 acts as an intracellular receptor to stimulate chemokine production and neutrophil recruitment in vivo. J. Exp. Med. 203, 203–213. 19. Park, J.H., Kim, Y.G., Shaw, M., Kanneganti, T.D., Fujimoto, Y., Fukase, K., Inohara, N., and Nunez, G. (2007). Nod1/RICK and TLR signaling regulate chemokine and antimicrobial innate immune responses in mesothelial cells. J. Immunol. 179, 514–521. 20. Chen, G.Y., Shaw, M.H., Redondo, G., and Nunez, G. (2008). The innate immune receptor Nod1 protects the intestine from inflammationinduced tumorigenesis. Cancer Res. 68, 10060–10067. 1Division of Hematology and Oncology, Department of Internal Medicine, 2University of Michigan Comprehensive Cancer Center, 3Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA. E-mail: [email protected]
DOI: 10.1016/j.cub.2008.12.027
cells, individual microtubules shift between these two states every 30 seconds or so, although the switches are stochastic. So, what governs the structure of the microtubule end and how can it shift from one structure to another? Each tubulin subunit is actually a dimer of two closely related proteins, a- and b-tubulin, which associate head-to-tail along microtubule protofilaments (Figure 1). The nucleotide status of b-tubulin determines the structure of the microtubule tip [3,4]. Growing microtubule ends are capped by GTP–tubulin subunits, which form straight protofilaments and maintain contacts between tubulins in neighboring protofilaments. The b-tubulin-bound GTP is hydrolyzed to GDP shortly after addition, meaning that the bulk of the microtubule is composed of GDP–tubulin subunits, which have a bent conformation but are held in the straight form by the cap of GTP–tubulin subunits [3,4]. Once the cap is lost, either through hydrolysis or dissociation, protofilaments of GDP–tubulin peel apart and depolymerize into subunits (Figure 1). Once the dimers depolymerize, the GDP bound to b-tubulin can exchange for GTP in solution to reform GTP–tubulin. Within the microtubule, only those b-tubulin subunits at the very tip can exchange their bound
Dispatch R175
A
B
Shorten
Length
nucleotide with nucleotide in solution [5], keeping the body of the microtubule composed of unstable GDP–tubulin subunits. Evidence supporting the GTP cap model for microtubule stability is strong, but to date we do not have direct evidence for the presence of such a cap in cellular microtubules. From studies of purified tubulins, we know that GTP–tubulin is the polymerizing subunit, that GTP hydrolysis lags slightly behind tubulin addition, and that the bulk of the microtubule is made up of GDP–tubulin. Measuring the size of the GTP–tubulin cap biochemically is difficult and requires both speed and manual dexterity [6]. Estimates for the size of the GTP cap vary, but it is likely to be between one and three tubulin layers deep at the microtubule tip [7], less than 1/400th of a short microtubule 10 mm in length. Given the difficulty of measuring GTP–tubulin caps in solutions of microtubules, it is not surprising that evidence for the GTP or GDP status of tubulin subunits in cellular microtubules is lacking. Now Dimitrov et al. [2] describe an antibody that is specific for the conformation of GTP–tubulin. Such an antibody would be difficult to generate by conventional immunization. Instead, the authors panned an antibody phage display library to find one that was specific for the conformation of GTP–tubulin. For antibody phage display, each phage expresses on its surface a single chain variable region (scFv) from an immunoglobin, while the DNA encoding the protein is contained within the phage [8]. Panning for scFvs that recognize microtubules assembled from tubulin–GTPgS, a non-hydrolyzable GTP analog, led to the isolation of sequences that recognize GTP–tubulin. Dimitrov et al. [2] then combined their scFv sequence with that for the Fc region of human IgG to create an antibody they call hMB11. The entire sequence was also fused to the fluorescent protein mCherry, creating a direct fluorescent tag. The hMB11 antibody appears to recognize the structure of tubulin–GTP, not simply GTP itself. Evidence for structure-specific binding comes from the fact that the antibody co-pelleted with microtubules assembled from tubulin bound to GMPCPP, another non-hydrolyzable GTP analog, but not to native microtubules assembled from tubulin-GTP subunits, nor to denatured
Rescue
Rescue
Grow
Time Current Biology
Figure 1. Microtubule assembly dynamics. (A) Schematic diagram of microtubule length changes over time. At steady state, microtubules continually convert between growing and shortening states. In a growing microtubule, the protofilaments — chains of tubulin dimers — are in a straight conformation, while these protofilaments bend away from the lattice and peel apart during shortening. The microtubule is held in a growing state by GTP–tubulin subunits at the tip (red circles). The core of the microtubule is composed of GDP–tubulin (blue circles). Loss of the GTP–tubulin cap leads to shortening. Remarkably, shortening microtubules can convert back to growth, a process called ‘rescue’, but the mechanism responsible for rescue is not known. (B) Dimitrov et al. [2] developed an antibody that recognizes the GTP-bound conformation of tubulin. This antibody shows remnants of GTP–tubulin within microtubules (red band), particularly in cells. These GTP– tubulin islands correlate with sites of rescue, suggesting that GTP–tubulin islands may stop the shortening process.
tubulins on immunoblots. The antibody also recognizes microtubules assembled in taxol, a microtubulestabilizing drug that prevents protofilaments from adopting the ‘peeling’ conformation [4]. While more evidence is needed to confirm that hMB11 recognizes a specific structure associated with GTP–tubulin, the initial results are impressive. Turning to cells, Dimitrov et al. [2] used their hMB11 antibody to localize GTP–tubulin within microtubules. Because the antibody recognizes a native structure, cells were examined without fixation, necessitating glycerol and low concentrations of taxol to render the microtubules stable during antibody incubation. hMB11 recognizes about 60% of microtubule ends, which fits reasonably well with estimates for the percentage of microtubules growing at any point in time. Surprisingly, hMB11 recognized not only the very tips of microtubules, but also spots located randomly along the microtubule lattice; the authors refer to these as ‘GTP remnants’. The presence of GTP–tubulin remnants within microtubules could contribute to rescue events by halting or slowing microtubule shortening and allowing microtubules to switch back to growth. To examine the relationship
between GTP–tubulin remnants and microtubule dynamics, Dimitrov et al. [2] recorded microtubule dynamic instability in cells expressing GFPtagged a-tubulin and then immediately lysed cells to localize hMB11-binding sites. Amazingly, hMB11 localized to sites where a rescue event had recently occurred, implying that internal GTP–tubulin remnants function to promote rescue events. Upon confirmation of the results presented in the study by Dimtrov et al. [2], the next big question will be to figure out how GTP–tubulin remnants end up in the microtubule lattice. These remnants were detectable to a lesser extent in microtubules assembled from purified tubulin, suggesting that whatever mechanism is responsible for the remnants, it is something intrinsic to tubulin/microtubules. Dimitrov et al. [2] propose that the remnants result from incomplete hydrolysis of all GTP– tubulin subunits in the microtubule lattice, leaving behind tiny areas where the subunits remain in the GTP-bound form. An alternative possibility is that the microtubule lattice can ‘breathe’, allowing subunits to enter and exit from the middle of the lattice. Such a ‘breathing’ model was originally proposed by Inoue [9] in order to explain microtubule turnover. While it
Current Biology Vol 19 No 4 R176
is well accepted that tubulin subunits are added and removed from polymer ends, it is possible that rare breathing events allow a few GTP–tubulin subunits into the middle of the microtubule lattice. Evidence for such exchange is lacking, although microtubules do display lattice defects [10], which could provide entry sites for GTP–tubulin. The hMB11 antibody described by Dimitrov et al. [2] adds to a growing list of new tools for the study of microtubule assembly. These tools are pushing experimental resolution to the nanometer scale [7,11], bringing experimental results closer to the level described by computational modeling [12], and providing the tools to unravel molecular events at microtubule ends that govern the assembly–disassembly
transitions unique to dynamic instability. References 1. Jordan, M.A., and Kamath, K. (2007). How do microtubule-targeted drugs work? An overview. Curr. Cancer Drug Targets 7, 325–334. 2. Dimitrov, A., Quesnoit, M., Moutel, S., Cantaloube, I., Pous, C., and Perez, F. (2008). Detection of GTP-tubulin conformation in vivo reveals a role for GTP remnants in microtubule rescues. Science 322, 1353–1356. 3. Howard, J., and Hyman, A.A. (2003). Dynamics and mechanics of the microtubule plus end. Nature 422, 753–758. 4. Downing, K.H. (2000). Structural basis for the interaction of tubulin with proteins and drugs that affect microtubule dynamics. Annu. Rev. Cell Dev. Biol. 16, 89–111. 5. Mitchison, T.J. (1993). Localization of an exchangeable GTP binding site at the plus ends of microtubules. Science 261, 1044–1047. 6. Caplow, M. (1992). Microtubule dynamics. Curr. Opin. Cell Biol. 4, 58–65. 7. Schek, H.T., III, Gardner, M.K., Cheng, J., Odde, D.J., and Hunt, A.J. (2007). Microtubule assembly dynamics at the nanometer scale. Curr. Biol. 17, 1445–1455.
8. Vaughan, T.J., Osborn, J.K., and Tempest, P.R. (1998). Human antibodies by design. Nat. Biotech. 16, 535–539. 9. Inoue, S. (1981). Cell division and the mitotic spindle. J. Cell Biol. 91, 131s–147s. 10. Chretien, D., Metoz, F., Verde, F., Karsenti, E., and Wade, R.H. (1992). Lattice defects in microtubules: protofilament numbers vary within individual microtubules. J. Cell Biol. 117, 1031–1040. 11. Kerssemakers, J.W.J., Munteanu, E.L., Laan, L., Noetzel, T., Janson, M.E., and Dogterom, M. (2006). Assembly dynamics of microtubules at molecular resolution. Nature 442, 709–712. 12. VanBuren, V., Cassimeris, L., and Odde, D.J. (2005). A mechanochemical model of microtubule structure and self-assembly kinetics. Biophys. J. 89, 2911–2926.
Department of Biological Sciences, 111 Research Drive, Lehigh University, Bethlehem, PA 18017, USA. E-mail: [email protected]
DOI: 10.1016/j.cub.2008.12.035
6.
7.
8.
9.
10.
11.
environmental factor that regulates fat storage. Proc. Natl. Acad. Sci. USA 101, 15718–15723. Rakoff-Nahoum, S., Paglino, J., EslamiVarzaneh, F., Edberg, S., and Medzhitov, R. (2004). Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118, 229–241. Mazmanian, S.K., Liu, C.H., Tzianabos, A.O., and Kasper, D.L. (2005). An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 122, 107–118. Stappenbeck, T.S., Hooper, L.V., and Gordon, J.I. (2002). Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells. Proc. Natl. Acad. Sci. USA 99, 15451–15455. Turnbaugh, P.J., Ley, R.E., Mahowald, M.A., Magrini, V., Mardis, E.R., and Gordon, J.I. (2006). An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027–1031. Rakoff-Nahoum, S., and Medzhitov, R. (2007). Regulation of spontaneous intestinal tumorigenesis through the adaptor protein MyD88. Science 317, 124–127. Wen, L., Ley, R.E., Volchkov, P.Y., Stranges, P.B., Avanesyan, L., Stonebraker, A.C., Hu, C., Wong, F.S., Szot, G.L., Bluestone, J.A., et al. (2008). Innate immunity and intestinal microbiota in the development of Type 1 diabetes. Nature 455, 1109–1113.
12. Chen, G., Shaw, M.H., Kim, Y.G., and Nunez, G. (2008). Nod-like receptors: Role in innate immunity and inflammatory disease. Annu. Rev. Pathol., epub ahead of print. 13. Akira, S., Uematsu, S., and Takeuchi, O. (2006). Pathogen recognition and innate immunity. Cell 124, 783–801. 14. Watanabe, T., Asano, N., Murray, P.J., Ozato, K., Tailor, P., Fuss, I.J., Kitani, A., and Strober, W. (2008). Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis. J. Clin. Invest. 118, 545–559. 15. Bouskra, D., Brezillon, C., Berard, M., Werts, C., Varona, R., Boneca, I.G., and Eberl, G. (2008). Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis. Nature 456, 507–510. 16. Hasegawa, M., Yang, K., Hashimoto, M., Park, J.H., Kim, Y.G., Fujimoto, Y., Nunez, G., Fukase, K., and Inohara, N. (2006). Differential release and distribution of Nod1 and Nod2 immunostimulatory molecules among bacterial species and environments. J. Biol. Chem. 281, 29054–29063. 17. Boughan, P.K., Argent, R.H., Body-Malapel, M., Park, J.H., Ewings, K.E., Bowie, A.G., Ong, S.J., Cook, S.J., Sorensen, O.E., Manzo, B.A., et al. (2006). Nucleotide-binding oligomerization domain-1 and epidermal growth factor receptor: critical regulators of beta-defensins during Helicobacter pylori infection. J. Biol. Chem. 281, 11637–11648.
Microtubule Assembly: Lattice GTP to the Rescue Recent work describes the surprising finding that cellular microtubules have islands of GTP-bound tubulin within their lattices, in contrast to the longstanding view that all but the very tips of growing microtubules are made up entirely of GDP–tubulin. These GTP–tubulin islands may act as stop signs or speed bumps, switching a shortening microtubule back into a growing state. Lynne Cassimeris Microtubule polymers are far from static; each polymer continually and unpredictably shifts between growing and shortening states (Figure 1). This turnover, a process called dynamic instability, allows microtubules to reorganize swiftly in response to cues. Dynamic turnover is critical for a number of cell functions and several anti-cancer drugs halt cells in the midst of division by blocking microtubule dynamics [1]. The most puzzling mechanistic aspects of dynamic instability are the switches between the polymerization and depolymerization states because these are such rare events compared with the addition and removal of thousands or tens of thousands of subunits from a microtubule end before a switch occurs. A recent paper in Science [2] provides surprising evidence that GTP-bound tubulin, the type of tubulin
dimer thought to be present only at microtubule ends, is also scattered throughout the microtubule lattice. These lattice GTP–tubulin subunits may function to stimulate rescue when a shortening microtubule stops losing subunits and begins to polymerize again. The structure of the microtubule tip governs whether the tubulin subunits that form the microtubule polymer will add or subtract from the polymer’s end (Figure 1). Polymerizing microtubules are typically not blunt-ended tubes, but instead have sheet-like extensions of tubulin protofilaments (Figure 1) that eventually close to form a tube. Shortening microtubules look very different — their protofilaments are no longer straight and they lose contact with their neighbors. These shortening protofilaments peel away from the microtubule lattice and soon fall apart into individual tubulin subunits. In
18. Masumoto, J., Yang, K., Varambally, S., Hasegawa, M., Tomlins, S.A., Qiu, S., Fujimoto, Y., Kawasaki, A., Foster, S.J., Horie, Y., et al. (2006). Nod1 acts as an intracellular receptor to stimulate chemokine production and neutrophil recruitment in vivo. J. Exp. Med. 203, 203–213. 19. Park, J.H., Kim, Y.G., Shaw, M., Kanneganti, T.D., Fujimoto, Y., Fukase, K., Inohara, N., and Nunez, G. (2007). Nod1/RICK and TLR signaling regulate chemokine and antimicrobial innate immune responses in mesothelial cells. J. Immunol. 179, 514–521. 20. Chen, G.Y., Shaw, M.H., Redondo, G., and Nunez, G. (2008). The innate immune receptor Nod1 protects the intestine from inflammationinduced tumorigenesis. Cancer Res. 68, 10060–10067. 1Division of Hematology and Oncology, Department of Internal Medicine, 2University of Michigan Comprehensive Cancer Center, 3Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA. E-mail: [email protected]
DOI: 10.1016/j.cub.2008.12.027
cells, individual microtubules shift between these two states every 30 seconds or so, although the switches are stochastic. So, what governs the structure of the microtubule end and how can it shift from one structure to another? Each tubulin subunit is actually a dimer of two closely related proteins, a- and b-tubulin, which associate head-to-tail along microtubule protofilaments (Figure 1). The nucleotide status of b-tubulin determines the structure of the microtubule tip [3,4]. Growing microtubule ends are capped by GTP–tubulin subunits, which form straight protofilaments and maintain contacts between tubulins in neighboring protofilaments. The b-tubulin-bound GTP is hydrolyzed to GDP shortly after addition, meaning that the bulk of the microtubule is composed of GDP–tubulin subunits, which have a bent conformation but are held in the straight form by the cap of GTP–tubulin subunits [3,4]. Once the cap is lost, either through hydrolysis or dissociation, protofilaments of GDP–tubulin peel apart and depolymerize into subunits (Figure 1). Once the dimers depolymerize, the GDP bound to b-tubulin can exchange for GTP in solution to reform GTP–tubulin. Within the microtubule, only those b-tubulin subunits at the very tip can exchange their bound
Dispatch R175
A
B
Shorten
Length
nucleotide with nucleotide in solution [5], keeping the body of the microtubule composed of unstable GDP–tubulin subunits. Evidence supporting the GTP cap model for microtubule stability is strong, but to date we do not have direct evidence for the presence of such a cap in cellular microtubules. From studies of purified tubulins, we know that GTP–tubulin is the polymerizing subunit, that GTP hydrolysis lags slightly behind tubulin addition, and that the bulk of the microtubule is made up of GDP–tubulin. Measuring the size of the GTP–tubulin cap biochemically is difficult and requires both speed and manual dexterity [6]. Estimates for the size of the GTP cap vary, but it is likely to be between one and three tubulin layers deep at the microtubule tip [7], less than 1/400th of a short microtubule 10 mm in length. Given the difficulty of measuring GTP–tubulin caps in solutions of microtubules, it is not surprising that evidence for the GTP or GDP status of tubulin subunits in cellular microtubules is lacking. Now Dimitrov et al. [2] describe an antibody that is specific for the conformation of GTP–tubulin. Such an antibody would be difficult to generate by conventional immunization. Instead, the authors panned an antibody phage display library to find one that was specific for the conformation of GTP–tubulin. For antibody phage display, each phage expresses on its surface a single chain variable region (scFv) from an immunoglobin, while the DNA encoding the protein is contained within the phage [8]. Panning for scFvs that recognize microtubules assembled from tubulin–GTPgS, a non-hydrolyzable GTP analog, led to the isolation of sequences that recognize GTP–tubulin. Dimitrov et al. [2] then combined their scFv sequence with that for the Fc region of human IgG to create an antibody they call hMB11. The entire sequence was also fused to the fluorescent protein mCherry, creating a direct fluorescent tag. The hMB11 antibody appears to recognize the structure of tubulin–GTP, not simply GTP itself. Evidence for structure-specific binding comes from the fact that the antibody co-pelleted with microtubules assembled from tubulin bound to GMPCPP, another non-hydrolyzable GTP analog, but not to native microtubules assembled from tubulin-GTP subunits, nor to denatured
Rescue
Rescue
Grow
Time Current Biology
Figure 1. Microtubule assembly dynamics. (A) Schematic diagram of microtubule length changes over time. At steady state, microtubules continually convert between growing and shortening states. In a growing microtubule, the protofilaments — chains of tubulin dimers — are in a straight conformation, while these protofilaments bend away from the lattice and peel apart during shortening. The microtubule is held in a growing state by GTP–tubulin subunits at the tip (red circles). The core of the microtubule is composed of GDP–tubulin (blue circles). Loss of the GTP–tubulin cap leads to shortening. Remarkably, shortening microtubules can convert back to growth, a process called ‘rescue’, but the mechanism responsible for rescue is not known. (B) Dimitrov et al. [2] developed an antibody that recognizes the GTP-bound conformation of tubulin. This antibody shows remnants of GTP–tubulin within microtubules (red band), particularly in cells. These GTP– tubulin islands correlate with sites of rescue, suggesting that GTP–tubulin islands may stop the shortening process.
tubulins on immunoblots. The antibody also recognizes microtubules assembled in taxol, a microtubulestabilizing drug that prevents protofilaments from adopting the ‘peeling’ conformation [4]. While more evidence is needed to confirm that hMB11 recognizes a specific structure associated with GTP–tubulin, the initial results are impressive. Turning to cells, Dimitrov et al. [2] used their hMB11 antibody to localize GTP–tubulin within microtubules. Because the antibody recognizes a native structure, cells were examined without fixation, necessitating glycerol and low concentrations of taxol to render the microtubules stable during antibody incubation. hMB11 recognizes about 60% of microtubule ends, which fits reasonably well with estimates for the percentage of microtubules growing at any point in time. Surprisingly, hMB11 recognized not only the very tips of microtubules, but also spots located randomly along the microtubule lattice; the authors refer to these as ‘GTP remnants’. The presence of GTP–tubulin remnants within microtubules could contribute to rescue events by halting or slowing microtubule shortening and allowing microtubules to switch back to growth. To examine the relationship
between GTP–tubulin remnants and microtubule dynamics, Dimitrov et al. [2] recorded microtubule dynamic instability in cells expressing GFPtagged a-tubulin and then immediately lysed cells to localize hMB11-binding sites. Amazingly, hMB11 localized to sites where a rescue event had recently occurred, implying that internal GTP–tubulin remnants function to promote rescue events. Upon confirmation of the results presented in the study by Dimtrov et al. [2], the next big question will be to figure out how GTP–tubulin remnants end up in the microtubule lattice. These remnants were detectable to a lesser extent in microtubules assembled from purified tubulin, suggesting that whatever mechanism is responsible for the remnants, it is something intrinsic to tubulin/microtubules. Dimitrov et al. [2] propose that the remnants result from incomplete hydrolysis of all GTP– tubulin subunits in the microtubule lattice, leaving behind tiny areas where the subunits remain in the GTP-bound form. An alternative possibility is that the microtubule lattice can ‘breathe’, allowing subunits to enter and exit from the middle of the lattice. Such a ‘breathing’ model was originally proposed by Inoue [9] in order to explain microtubule turnover. While it
Current Biology Vol 19 No 4 R176
is well accepted that tubulin subunits are added and removed from polymer ends, it is possible that rare breathing events allow a few GTP–tubulin subunits into the middle of the microtubule lattice. Evidence for such exchange is lacking, although microtubules do display lattice defects [10], which could provide entry sites for GTP–tubulin. The hMB11 antibody described by Dimitrov et al. [2] adds to a growing list of new tools for the study of microtubule assembly. These tools are pushing experimental resolution to the nanometer scale [7,11], bringing experimental results closer to the level described by computational modeling [12], and providing the tools to unravel molecular events at microtubule ends that govern the assembly–disassembly
transitions unique to dynamic instability. References 1. Jordan, M.A., and Kamath, K. (2007). How do microtubule-targeted drugs work? An overview. Curr. Cancer Drug Targets 7, 325–334. 2. Dimitrov, A., Quesnoit, M., Moutel, S., Cantaloube, I., Pous, C., and Perez, F. (2008). Detection of GTP-tubulin conformation in vivo reveals a role for GTP remnants in microtubule rescues. Science 322, 1353–1356. 3. Howard, J., and Hyman, A.A. (2003). Dynamics and mechanics of the microtubule plus end. Nature 422, 753–758. 4. Downing, K.H. (2000). Structural basis for the interaction of tubulin with proteins and drugs that affect microtubule dynamics. Annu. Rev. Cell Dev. Biol. 16, 89–111. 5. Mitchison, T.J. (1993). Localization of an exchangeable GTP binding site at the plus ends of microtubules. Science 261, 1044–1047. 6. Caplow, M. (1992). Microtubule dynamics. Curr. Opin. Cell Biol. 4, 58–65. 7. Schek, H.T., III, Gardner, M.K., Cheng, J., Odde, D.J., and Hunt, A.J. (2007). Microtubule assembly dynamics at the nanometer scale. Curr. Biol. 17, 1445–1455.
8. Vaughan, T.J., Osborn, J.K., and Tempest, P.R. (1998). Human antibodies by design. Nat. Biotech. 16, 535–539. 9. Inoue, S. (1981). Cell division and the mitotic spindle. J. Cell Biol. 91, 131s–147s. 10. Chretien, D., Metoz, F., Verde, F., Karsenti, E., and Wade, R.H. (1992). Lattice defects in microtubules: protofilament numbers vary within individual microtubules. J. Cell Biol. 117, 1031–1040. 11. Kerssemakers, J.W.J., Munteanu, E.L., Laan, L., Noetzel, T., Janson, M.E., and Dogterom, M. (2006). Assembly dynamics of microtubules at molecular resolution. Nature 442, 709–712. 12. VanBuren, V., Cassimeris, L., and Odde, D.J. (2005). A mechanochemical model of microtubule structure and self-assembly kinetics. Biophys. J. 89, 2911–2926.
Department of Biological Sciences, 111 Research Drive, Lehigh University, Bethlehem, PA 18017, USA. E-mail: [email protected]
DOI: 10.1016/j.cub.2008.12.035